The overall goal of this research is to elucidate the catalytic mechanism(s) of enzymes catalyzing a PLP-dependent beta-elimination reaction. Studies will be carried out to determine the kinetic mechanism, location of rate-limiting steps along the reaction coordinate, and the chemistry taking place during substrate turnover in the active site of the enzyme. Experimental techniques available to acquire this information include initial rate studies in the absence and presence of products or dead-end inhibitors, pH and isotope effect studies. The main purpose is delineation of the reaction coordination with rate constants for individual steps, and elucidation of the structure of the transition-state and of any intermediates. Information on the chemical mechanism can be deduced from the pH dependence of the kinetic parameters (V, V/K, and the Ki for inhibitors and substrates), and from primary and secondary isotope effects. This proposal may be divided into four parts. First, determination of the kinetic mechanism of the reverse reaction for E. coli tryptophan indole- lyase. The appropriate Haldane equations relating the equilibrium constant (which will be measured independently) to the kinetic parameters will be utilized to check for internal consistency of the mechanism. Also, pH and isotope effect studies with slow alternate substrates for this enzyme will be undertaken. Second, measurement of the secondary deuterium isotope effects for tryptophan indole-lyase, and the primary and secondary deuterium isotope effect for tryptophan indole-lyase and both tyrosine phenol-lyases from C. freundii and E. herbicola with S-methyl-L cysteine, and determination of these effects in D2O. Third, determination of the solvent deuterium isotope effects for both tyrosine phenol-lyases, and in the reverse direction for tryptophan indole-lyase. Proton inventories will be determined for the above enzymes. Fourth, measurement of the kinetic parameters and primary deuterium isotope effects and pH studies to determine the catalytic mechanism of the PLP-dependent enzyme, O- phosphorylethanolamine phospho-lyase. There is virtually no mechanistic data on this enzyme which catalyzes the PLP-dependent hydrolysis of O- phosphorylethanolamine (PEA) to acetaldehyde, ammonia, and orthophosphate. Knowledge of he catalytic mechanism for PEA phospho-lyase will further our overall understanding of phosphoryl transfer mechanisms as this enzymes mechanism appears to be unique in that regard. Also, this study of enzyme systems catalyzing the same type of reaction will provide the first in- depth comparison of three catalytic mechanisms through the systematic use of multiple isotope effects (primary and secondary deuterium and solvent deuterium). The knowledge derived for the study should broaden our understanding and interpret solvent deuterium isotope effects.